Rubicon (RUN domain protein as Beclin1 interacting and cysteine-rich containing) protein has recently been identified as a novel Beclin1-binding autophagy protein wherein it negatively regulates the vesicle maturation step of autophagy and endocytic pathway. Our preliminary study has revealed for the first time the direct crosstalk between autophagy and phagocytosis machineries by demonstrating that Rubicon autophagy protein is an essential positive regulator of the NADPH oxidase complex, inducing ROS production upon microbial infection or plasma membrane Toll-like receptor (TLR) activation. While Rubicon primarily associates with the Beclin1-Vps34 autophagy complex and suppresses autophagosome maturation step under normal conditions, it is rapidly recruited to the p22phox-gp91phox NADPH oxidase complex upon microbial infection, facilitating the stabilization and phagosomal translocation of the NADPH oxidase complex to induce a ROS burst, inflammatory cytokine production, and thereby, potent anti-microbial activities. After the completion of this cycle, Rubicon then returns to the Beclin1-Vps34 autophagic complex in preparation for the next round of anti-microbial activity. Remarkably, Rubicon's actions in the Beclin1-Vps34 autophagy complex and in the p22phox-gp91phox NADPH oxidase complex are functionally and genetically separable. Based on these results, we hypothesize that Rubicon orchestrates two ancient innate immune machineries, autophagy and phagocytosis, toggling between the two depending on the environmental stimuli, ultimately generating an optimal intracellular immune milieu against microbial infection. This proposal is highly novel and innovative, and a successful outcome will potentially lead to a paradigm shift in our understanding of host ancient innate immunity and the development of anti-microbe strategies. PUBLIC HEALTH RELEVANCE: Phagocytes such as neutrophils and monocytes play an essential role in host defenses against microbial pathogens. Specifically, the phagocytic NADPH oxidase is recognized as a critical component of innate immunity as it is responsible for the generation of microbicidal reactive oxygen species (ROS). Additionally, autophagy, a cytoprotective mechanism involving the formation of double-membrane vesicles called autophagosomes, sequesters cytoplasmic damaged organelles, protein aggregates, or invading pathogens for degradation. Furthermore, autophagy facilitates phagocytosis by promoting phagosome maturation and rapid acidification, and by preventing pathogens from escaping into the cytosol, but how autophagy couples with phagocytosis remains elusive. The main goal of this proposal is to elucidate the crosstalk between phagocytosis and autophagy in building an effective intracellular innate immune milieu against microbial pathogens.